A Neutralizing RNA Aptamer against EGFR Causes Selective Apoptotic Cell Death

Nucleic acid aptamers have been developed as high-affinity ligands that may act as antagonists of disease-associated proteins. Aptamers are non immunogenic and characterised by high specificity and low toxicity thus representing a valid alternative to antibodies or soluble ligand receptor traps/decoys to target specific cancer cell surface proteins in clinical diagnosis and therapy. The epidermal growth factor receptor (EGFR) has been implicated in the development of a wide range of human cancers including breast, glioma and lung. The observation that its inhibition can interfere with the growth of such tumors has led to the design of new drugs including monoclonal antibodies and tyrosine kinase inhibitors currently used in clinic. However, some of these molecules can result in toxicity and acquired resistance, hence the need to develop novel kinds of EGFR-targeting drugs with high specificity and low toxicity. Here we generated, by a cell-Systematic Evolution of Ligands by EXponential enrichment (SELEX) approach, a nuclease resistant RNA-aptamer that specifically binds to EGFR with a binding constant of 10 nM. When applied to EGFR-expressing cancer cells the aptamer inhibits EGFR-mediated signal pathways causing selective cell death. Furthermore, at low doses it induces apoptosis even of cells that are resistant to the most frequently used EGFR-inhibitors, such as gefitinib and cetuximab, and inhibits tumor growth in a mouse xenograft model of human non-small-cell lung cancer (NSCLC). Interestingly, combined treatment with cetuximab and the aptamer shows clear synergy in inducing apoptosis in vitro and in vivo. In conclusion, we demonstrate that this neutralizing RNA-aptamer is a promising bio-molecule that can be developed as a more effective alternative to the repertoire of already existing EGFR-inhibitors

Effects of elevated seawater pCO2 on gene expression patterns in the gills of the green crab, Carcinus maenas

Background: The green crab Carcinus maenas is known for its high acclimation potential to varying environmental abiotic conditions. A high ability for ion and acid-base regulation is mainly based on an efficient regulation apparatus located in gill epithelia. However, at present it is neither known which ion transport proteins play a key role in the acid-base compensation response nor how gill epithelia respond to elevated seawater pCO2 as predicted for the future. In order to promote our understanding of the responses of green crab acid-base regulatory epithelia to high pCO2, Baltic Sea green crabs were exposed to a pCO2 of 400 Pa. Gills were screened for differentially expressed gene transcripts using a 4,462-feature microarray and quantitative real-time PCR. Results: Crabs responded mainly through fine scale adjustment of gene expression to elevated pCO2. However, 2% of all investigated transcripts were significantly regulated 1.3 to 2.2-fold upon one-week exposure to CO2 stress. Most of the genes known to code for proteins involved in osmo- and acid-base regulation, as well as cellular stress response, were were not impacted by elevated pCO2. However, after one week of exposure, significant changes were detected in a calcium-activated chloride channel, a hyperpolarization activated nucleotide-gated potassium channel, a tetraspanin, and an integrin. Furthermore, a putative syntaxin-binding protein, a protein of the transmembrane 9 superfamily, and a Cl-/HCO3 - exchanger of the SLC 4 family were differentially regulated. These genes were also affected in a previously published hypoosmotic acclimation response study. Conclusions: The moderate, but specific response of C. maenas gill gene expression indicates that (1) seawater acidification does not act as a strong stressor on the cellular level in gill epithelia; (2) the response to hypercapnia is to some degree comparable to a hypoosmotic acclimation response; (3) the specialization of each of the posterior gill arches might go beyond what has been demonstrated up to date; and (4) a re-configuration of gill epithelia might occur in response to hypercapnia

The creatine kinase system and pleiotropic effects of creatine

The pleiotropic effects of creatine (Cr) are based mostly on the functions of the enzyme creatine kinase (CK) and its high-energy product phosphocreatine (PCr). Multidisciplinary studies have established molecular, cellular, organ and somatic functions of the CK/PCr system, in particular for cells and tissues with high and intermittent energy fluctuations. These studies include tissue-specific expression and subcellular localization of CK isoforms, high-resolution molecular structures and structure–function relationships, transgenic CK abrogation and reverse genetic approaches. Three energy-related physiological principles emerge, namely that the CK/PCr systems functions as (a) an immediately available temporal energy buffer, (b) a spatial energy buffer or intracellular energy transport system (the CK/PCr energy shuttle or circuit) and (c) a metabolic regulator. The CK/PCr energy shuttle connects sites of ATP production (glycolysis and mitochondrial oxidative phosphorylation) with subcellular sites of ATP utilization (ATPases). Thus, diffusion limitations of ADP and ATP are overcome by PCr/Cr shuttling, as most clearly seen in polar cells such as spermatozoa, retina photoreceptor cells and sensory hair bundles of the inner ear. The CK/PCr system relies on the close exchange of substrates and products between CK isoforms and ATP-generating or -consuming processes. Mitochondrial CK in the mitochondrial outer compartment, for example, is tightly coupled to ATP export via adenine nucleotide transporter or carrier (ANT) and thus ATP-synthesis and respiratory chain activity, releasing PCr into the cytosol. This coupling also reduces formation of reactive oxygen species (ROS) and inhibits mitochondrial permeability transition, an early event in apoptosis. Cr itself may also act as a direct and/or indirect anti-oxidant, while PCr can interact with and protect cellular membranes. Collectively, these factors may well explain the beneficial effects of Cr supplementation. The stimulating effects of Cr for muscle and bone growth and maintenance, and especially in neuroprotection, are now recognized and the first clinical studies are underway. Novel socio-economically relevant applications of Cr supplementation are emerging, e.g. for senior people, intensive care units and dialysis patients, who are notoriously Cr-depleted. Also, Cr will likely be beneficial for the healthy development of premature infants, who after separation from the placenta depend on external Cr. Cr supplementation of pregnant and lactating women, as well as of babies and infants are likely to be of benefit for child development. Last but not least, Cr harbours a global ecological potential as an additive for animal feed, replacing meat- and fish meal for animal (poultry and swine) and fish aqua farming. This may help to alleviate human starvation and at the same time prevent over-fishing of oceans

In Vivo Ectopic Implantation Model to Assess Human Mesenchymal Progenitor Cell Potential

Clinical interest on human mesenchymal progenitor cells (hMPC) relies on their potential applicability in cell-based therapies. An in vitro characterization is usually performed in order to define MPC potency. However, in vitro predictions not always correlate with in vivo results and thus there is no consensus in how to really assess cell potency. Our goal was to provide an in vivo testing method to define cell behavior before therapeutic usage, especially for bone tissue engineering applications. In this context, we wondered whether bone marrow stromal cells (hBMSC) would proceed in an osteogenic microenvironment. Based on previous approaches, we developed a fibrin/ceramic/BMP-2/hBMSCs compound. We implanted the compound during only 2 weeks in NOD-SCID mice, either orthotopically to assess its osteoinductive property or subcutaneously to analyze its adequacy as a cell potency testing method. Using fluorescent cell labeling and immunohistochemistry techniques, we could ascertain cell differentiation to bone, bone marrow, cartilage, adipocyte and fibrous tissue. We observed differences in cell potential among different batches of hBMSCs, which did not strictly correlate with in vitro analyses. Our data indicate that the method we have developed is reliable, rapid and reproducible to define cell potency, and may be useful for testing cells destined to bone tissue engineering purposes. Additionally, results obtained with hMPCs from other sources indicate that our method is suitable for testing any potentially implantable mesenchymal cell. Finally, we propose that this model could successfully be employed for bone marrow niche and bone tumor studies. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12015-013-9464-1) contains supplementary material, which is available to authorized users

Floral diversity during Plio-Pleistocene Siwalik sedimentation (Kimin Formation) in Arunachal Pradesh, India, and its palaeoclimatic significance

A morpho-taxonomic study of leaf remains from the upper part of the Siwalik succession of sediments (Kimin Formation; upper Pliocene to lower Pleistocene) of Papumpare district, Arunachal Pradesh, India, revealed 23 species representing 20 genera belonging to 15 angiosperm families. The recovered fossil leaves are comparable to modern Bambusa tulda Roxb. (Poaceae), Mangifera indica Linn., Dracontomelum mangiferum Blume (Anacardiaceae); Chonemorpha macrophylla G. Don (Apocynaceae); Pongamia pinnata (L) Pierre., Millettia pachycarpa Benth., Dalbergia rimosa Roxb., Millettia extensa (Fabaceae); Macaranga denticulate Muell. Arg., Croton caudatus Geisel. (Euphorbiaceae); Combretum decandrum Roxb. (Combretaceae); Dysoxylum procerum Hiern. (Meliaceae); Dipterocarpus sp. Gaertn.f. (Dipterocarpaceae); Actinodaphne angustifolia Nees., Actinodaphne obovata Blume., Lindera pulcherrima Benth., Litsea salicifolia Roxb. (Lauraceae); Calophyllum polyanthum Wall. (Clusiaceae); Knema glaucescens Hook.f. (Myristaceae); Canarium bengalense Roxb. (Burseraceae); Quercus lamellose Smith; Quercus semicarpifolia Smith (Fagaceae); and Berchemia floribunda Wall. (Rhamnaceae). Among these taxa, 11 species are recorded as new to the Neogene flora of India. Analysis of the floral assemblage with respect to the distribution pattern of modern equivalent taxa and the physiognomic characters of the fossil leaves, suggests that a tropical evergreen forest was growing in a warm humid climate in the region at the time of deposition. This is in contrast to modern tropical semi-evergreen forests that occupy the area. Values of mean annual temperature (MAT) of 29.3°C and mean annual precipitation (MAP) of 290 mm have been calculated using leaf-margin characters and fossil leaf size